Abstract Synthetic lethal approaches for `personalized' therapy of specific cancer subsets is a strategy that can attack the initial drivers of genetic instability, possibly eliminating tumor heterogeneity, as well as the emergence of resistant and metastatic cancer cells. We recently discovered that cancer cells that lost one allele of the RPRD1B/Kub5-Hera (K-H) gene, corresponding to ~50+% loss of protein, exhibit atypical elevations of PARP1 activities and a ?BRCAness? phenotype in BRCA-proficient cancer cells. We hypothesize that K-H binds to the C-terminal domain (CTD) of RNA polymerase II (RNAPII) to preferentially direct transcription of genes containing sequence-specific CHR-motif promoters, most importantly cyclin-dependent kinase 1 (CDK1). CDK1, in turn, stimulates BRCA1 phosphorylation and HR function. K-H loss in breast or nonsmall cell lung cancers (NSCLC), by copy number variation (CNV), mRNA expression or specific SNPs in patient tumors, results in a unique `cancer vulnerability of persistent R-loop formation and HR deficiency', whereby cells are dependent on PARP1- driven alternative non-homologous end joining (alt-NHEJ) repair. While this drives genetic instability due to elevated R-loops, a BRCA deficiency and a dependency on error-prone alt-NHEJ, the `vulnerability in genetic instability' can be exploited using PARP inhibitors (PARPis) that block redundant DSB repair and cause lethality during replication. We will complete two specific aims: Aim 1: To perform structure/function analyses using rationally-derived K-H mutation(s) and patient- derived tumor SNPs to delineate the mechanism by which aberrant K-H mutations alter PARP1 activity and CDK1 levels that, in turn, affect downstream BRCA1-HR function and cellular responses to PARP1is or IR. (Years 1-5). Aim 2: To optimize antitumor efficacy of breast or NSCLC cancer xenografts expressing mutant or loss of K-H protein expression using clinically-relevant PARPis or IR (Years 1-5). Completion of the proposed research will define the mechanism by which K-H directs RNAPII-driven CHR motif-specific gene expression and CDK1-BRCA1 HR function. In turn, understanding this mechanism will allow exploitation of the consequences of K-H loss and optimization of synthetic lethal approaches using PARPis or IR for treatment of specific cancer subsets. The studies will also reveal additional, exploitable cancer vulnerabilities. Our studies are novel as they define how a RNA transcription termination factor can direct transcription and DNA repair, revealing exploitable cancer vulnerabilities that can be translated.